Seed-Preferred Regulatory Elements

ABSTRACT

The present invention provides compositions and methods for regulating expression of nucleotide sequences in a plant. Compositions are novel nucleotide sequences for a tissue preferred promoter isolated from the sorghum 16 kDa oleosin coding region. The sequences drive expression preferentially to seed tissue, and most preferably to embryo and/or aleurone tissue of a plant. Functional fragments of same are also provided. A method for expressing a nucleotide sequence in a plant using the regulatory sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS-REFERENCE PARAGRAPH

This application is a continuation of U.S. patent application Ser. No.12/145,560 filed Jun. 25, 2008 which claims the benefit of U.S.Provisional Application No. 60/955,409, filed Aug. 13, 2007, which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Expression of DNA sequences in a plant host is dependent upon thepresence of regulatory elements that are functional within the planthost. Choice of the regulatory element will determine when and wherewithin the organism the DNA sequence is expressed. Where continuousexpression is desired throughout the cells of a plant, and/or throughoutdevelopment, constitutive promoters are utilized. In contrast, wheregene expression in response to a stimulus is desired, induciblepromoters are the regulatory element of choice. Where expression inspecific tissues or organs are desired, tissue-preferred promoters canbe used. That is, they may drive expression in particular tissues ororgans. With any of these variables, additional regulatory sequencesupstream and/or downstream from a core promoter sequence may be includedin expression constructs of transformation vectors to bring aboutvarying levels of expression of nucleotide sequences in a transgenicplant.

As this field develops and more genes become accessible, a greater needexists for transformed plants with multiple genes. These multipleexogenous genes typically need to be controlled by separate regulatorysequences however. Further, some genes should be regulatedconstitutively whereas other genes should be expressed at certaindevelopmental stages or locations in the transgenic organism.Accordingly, a variety of regulatory sequences having diverse effects isneeded.

Diverse regulatory sequences are also needed as undesirable biochemicalinteractions can result from using the same regulatory sequence tocontrol more than one gene. Transformation with multiple copies of aregulatory element may cause problems, such that expression of one ormore genes may be affected.

Isolation and characterization of a promoter that can serve as aregulatory element for expression of isolated nucleotide sequences ofinterest are needed for influencing various traits in plants. Theinventors have isolated just such a promoter.

SUMMARY OF THE INVENTION

The invention is directed to a promoter from a Sorghum bicoloroleosin-encoding gene, useful as a regulatory region and providing forexpression of a nucleotide sequence of interest. In an embodiment theexpression is driven in a seed-embryo/aleurone preferred manner. Theinvention is further directed to functional fragments which function isto drive expression of a nucleotide sequence of interest in aseed-embryo/aleurone preferred manner. Expression cassettes having thepromoter driving expression of a nucleotide sequence, plants expressingsame, and methods of use in modulating expression of nucleotidessequences are within the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

The seed of monocot plants include a seed coat, the embryo, and a supplyof stored food, the endosperm. Cereal endosperm mature seed includesthree cell types: the aleurone cells, the starchy endosperm, and thebasal endosperm transfer cells. The aleurone is a layer of denselycytoplasmic cells covering the surface of the endosperm, just beneaththe maternal pericarp tissue. When the seed germinates, the aleuronecells are stimulated to secrete enzymes that break down the storagecompounds in the endopersm, allowing amino acids and sugars to becomeavailable to the growing seedling, which develops from the embryo.

In accordance with the invention, nucleotide sequences are provided thatallow regulation of transcription in a seed-embroy/aleurone preferredmanner. Thus, the compositions of the present invention comprise novelnucleotide sequences for plant regulatory elements natively associatedwith the nucleotide sequences coding for Sorghum bicolor oleosinprotein, identified here as SB-OLE.

Oleosins are 16 kd to 24 kd structural proteins on the surface ofintracellular oil bodies in seeds. Oleosins in maize have been studiedand three have been categorized by the molecular weight of the encodedprotein: OLE16, OLE17 and OLE18. OLE16 in maize is a 16 kd oleosin,considered a low molecular weight oleosin compared to the 17 kd and 18kd oleosins. OLE16 has been mapped to a locus on chromosome 2 (near theb1 gene). Lee and Huang, (1994) “Genes encoding oleosins in maize kernelof inbreds Mo17 and B73” Plant Mol. Biol. 26(6):1981-1987.

In an embodiment, the regulatory element drives transcription in aseed-preferred manner, and in particular to the embryo and/or aleuronetissue of seed, wherein said regulatory element comprises a nucleotidesequence selected from the group consisting of: a) sequences nativelyassociated with, and that regulate expression of DNA coding for sorghumSB-OLE (Sorghum bicolor oleosin protein); or b) SEQ ID NO: 1; c) SEQ IDNO: 2; or d) a sequence comprising a functional fragment of thenucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

Further embodiments are to expression cassettes, transformation vectors,plants, and plant cells comprising the above nucleotide sequences. Theinvention is further to methods of using the sequence in plants andplant cells.

A method for expressing an isolated nucleotide sequence in a plant usingthe regulatory sequences disclosed herein is provided. The methodcomprises transforming a plant cell with a transformation vector thatcomprises an isolated nucleotide sequence linked to one or more of theplant regulatory sequences of the present invention and regenerating astably transformed plant from the transformed plant cell. In thismanner, the regulatory sequences are useful for controlling theexpression of endogenous as well as exogenous products in aseed-preferred, and in an embryo and aleurone tissue-preferred manner.

Alternatively, it can be desirable to inhibit expression of a native DNAsequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished, for example, withtransformation of the plant to comprise the tissue-specific promoterlinked to an antisense nucleotide sequence, hairpin, RNA interfering orother nucleic acid molecule, such that tissue-preferred expression ofthe molecule interferes with translation of the mRNA of the native DNAsequence or otherwise inhibits expression of same in a subset of theplant's cells.

Under the regulation of the regulatory element will be a sequence ofinterest, which will provide for modification of the phenotype of theplant. Such modification includes modulating the production of anendogenous product, as to amount, relative distribution, or the like, orproduction of an exogenous expression product to provide for a novelfunction or product in the plant. Such a promoter is useful for avariety of applications, such as production of transgenic plants andseed with desired seed traits, including, for example, altered oilcontent, protein quality, cell growth or nutrient quality.

By “tissue-preferred” promoter is intended expression which is capableof transcribing a nucleotide sequence efficiently and expressing saidsequence at modulated levels in the described tissues, here the seedtissues, and in an embodiment the embryo an/or aleurone cells, than inother tissues of the plant. Tissue can refer to a cell of a particulartissue.

By “regulatory element” is intended sequences responsible for expressionof the linked nucleic acid molecule including, but not limited to,promoters, terminators, enhancers, introns, and the like.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto. It usuallycomprises a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements which impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ region upstream from theparticular promoter region. Thus the promoter region disclosed here maycomprise upstream regulatory elements such as those responsible fortissue and temporal expression of the nucleic acid molecule, and mayinclude enhancers, the DNA response element for a transcriptionalregulatory protein, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, activatorsequence and the like.

In the same manner, the promoter elements which enable such expressioncan be identified, isolated, and used with other core promoters toconfer seed-preferred expression or embryo- and/or aleurone-preferredexpression. By core promoter is meant the minimal sequence required toinitiate transcription, such as the sequence called the TATA box whichis common to promoters in genes encoding proteins. Thus the upstreampromoter of SB-OLE can optionally be used in conjunction with its own,or core promoters from other sources. The promoter may be native ornon-native to the cell in which it is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to drive tissue preferred expression retained; i.e., functionalfragments.

It is recognized that expression levels of mRNA can be modulated withspecific deletions of portions of the promoter sequence. Thus, thepromoter can be modified to be a weak or strong promoter. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. Generally, atleast about 20 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence of interest.

It is recognized that to increase transcription levels enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon.

The term “isolated” refers to material, such as a nucleic acid orprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment, or (2) if the material is in itsnatural environment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art. One method is the use of primers andgenomic DNA used in conjunction with the Genome Walker Kit™ (Clonetech).

The SB-OLE promoter set forth in SEQ ID NO: 1 is 948 nucleotides inlength. The SB-OLE promoter was isolated by identifying the Sorghumbicolor ortholog to the maize 16 KD oleosin, then identifying upstreamsequence that was available from the publicly available Genome SurveySequence (GSS) collection.

The regulatory regions of the invention may be isolated from any plant,including, but not limited to sorghum (Sorghum bicolor, Sorghumvulgare), corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean(Glycine max), tobacco (Nicotiana tabacum), millet (Panicum spp.),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), oats (Avena sativa), barley (Hordeum vulgare),vegetables, ornamentals, and conifers. Preferably, plants include corn,soybean, sunflower, safflower, canola, wheat, barley, rye, alfalfa, andsorghum.

Promoters isolated from one plant species can be expected to express inother plant species. For example, maize promoters have been usedrepeatedly to drive expression of genes in non-maize plants, includingtobacco (Yang et al. (1990) “Maize sucrose synthase-1 promoter drivesphloem cell-specific expression of GUS gene in transgenic tobaccoplants” Proc. Natl. Acad. Sci. USA 87:4144-4148; Geffers et al., (2000)“Anaerobiosis-specific interaction of tobacco nuclear factors withcis-regulatory sequences in the maize GapC4 promoter” Plant Mol. Biol.43:11-21); cultured rice cells (Vilardell et al., (1991) “Regulation ofthe maize rab 17 gene promoter in transgenic heterologous systems” PlantMol. Biol. 17:985-993), wheat (Oldach et al., (2001) “Heterologousexpression of genes mediating enhanced fungal resistance in transgenicwheat” Mol. Plant. Microbe Interact. 14:832-838; Brinch-Pedersen et al.,(2003) “Concerted action of endogenous and heterologous phytase onphytic acid degradation in seed of transgenic wheat (Triticum aestivumL.)” Transgenic Res. 12:649-659), rice (Cornejo et al., (1993) “Activityof a maize ubiquitin promoter in transgenic rice” Plant Mol. Biol.23:567-581; Takimoto et al., (1994) “Non-systemic expression of astress-response maize polyubiquitin gene (Ubi-1) in transgenic riceplants” Plant Mol. Biol. 26:1007-1012), sunflower (Roussell et al.,(1988) “Deletion of DNA sequences flanking an Mr 19,000 zein genereduces its transcriptional activity in heterologous plant tissues” Mol.Gen. Genet. 211:202-209), and protoplasts of carrot (Roussell et al.,1988).

Regulatory sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the codingregion (i.e.: orthologs) of the sequences set forth herein. In thesetechniques, all or part of the known coding sequence is used as a probewhich selectively hybridizes to other sequences present in a populationof cloned genomic DNA fragments (i.e. genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocolsin Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

An orthologous gene is a gene from a different species that encodes aproduct having the same or similar function, e.g., catalyzing the samereaction as a product encoded by a gene from a reference organism. Thus,an ortholog includes polypeptides having less than, e.g., 50% amino acidsequence identity, but which ortholog encodes a polypeptide having thesame or similar function. Databases such GenBank may be employed toidentify sequences related to the Sorghum bicolor, for example.Alternatively, recombinant DNA techniques such as hybridization or PCRmay be employed to identify sequences related to the sorghum oleosinsequences or to clone the equivalent sequences from different DNAs.

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” is a regulatory sequence variantformed by one or more deletions from a larger regulatory element. Forexample, the 5′ portion of a promoter up to the TATA box near thetranscription start site can be deleted without abolishing promoteractivity, as described by Opsahl-Sorteberg et al., “Identification of a49-bp fragment of the HvLTP2 promoter directing aleruone cell specificexpression” Gene 341:49-58 (2004). Such fragments should retain promoteractivity, particularly the ability to drive expression in the selecttissue. Activity can be measured by Northern blot analysis, reporteractivity measurements when using transcriptional fusions, and the like.See, for example, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology See particularly, Mullis et al. (1987) Methods Enzymol.155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, NewYork).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al. (1990) PCR Protocols, A Guideto Methods and Applications, eds., Academic Press). Primers used inisolating the promoter of the present invention are shown below.

The regulatory elements disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant when operably linked with an isolated nucleotide sequenceof interest whose expression is to be controlled to achieve a desiredphenotype.

By “operably linked” is intended a functional linkage between aregulatory region and a second sequence, wherein the regulatory sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence. The expression cassette can include theregulatory sequences of the invention operably linked to at least onesequence of interest.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat. No. 6,187,994. The term “nucleotide sequence of interest”refers to a nucleic acid molecule (which may also be referred to as apolynucleotide) which can be an RNA molecule as well as DNA molecule,and can be a molecule that encodes for a desired polypeptide or protein,but also may refer to nucleic acid molecules that do not constitute anentire gene, and which do not necessarily encode a polypeptide orprotein. For example, when used in a homologous recombination process,the promoter may be placed in a construct with a sequence that targetsan area of the chromosome in the plant but may not encode a protein. Ifdesired, the nucleotide sequence of interest can be optimized for planttranslation by optimizing the codons used for plants and the sequencearound the translational start site for plants. Sequences resulting inpotential mRNA instability can also be avoided.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated nucleotide sequences ofinterest for expression in the desired plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence of interest under the transcriptional controlof the regulatory elements. Alternatively, a specific result can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors in the plant.This down regulation can be achieved through many different approachesknown to one skilled in the art, including antisense, cosupression, useof hairpin formations, or others, and discussed infra. Importation orexportation of a cofactor also allows for control of plant composition.It is recognized that the regulatory elements may be used with theirnative or other coding sequences to increase or decrease expression ofan operably linked sequence in the transformed plant or seed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, and grain characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors, and hormones from plants and othereukaryotes as well as prokaryotic organisms.

Modifications that affect grain traits include increasing the content ofoleic acid, or altering levels of saturated and unsaturated fatty acids.Likewise, the level of plant proteins, particularly modified proteinsthat improve the nutrient value of the plant, can be increased. This isachieved by the expression of such proteins having enhanced amino acidcontent.

Increasing the levels of lysine and sulfur-containing amino acids may bedesired as well as the modification of starch type and content in theseed. Hordothionin protein modifications are described in WO 9416078filed Apr. 10, 1997; WO 9638562 filed Mar. 26, 1997; WO 9638563 filedMar. 26, 1997 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997. Anotherexample is lysine and/or sulfur-rich plant protein encoded by thesoybean 2S albumin described in WO 9735023 filed Mar. 20, 1996, and thechymotrypsin inhibitor from barley, Williamson et al. (1987) Eur. J.Biochem. 165:99-106.

Agronomic traits in plants can be improved by altering expression ofgenes that: affect the response of plant growth and development duringenvironmental stress, Cheikh-N et al. (1994) Plant Physiol.106(1):45-51) and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier et al. (1995) Plant Physiol.107(2):385-391.

It is recognized that any nucleotide sequence of interest, including thenative coding sequence, can be operably linked to the regulatoryelements of the invention and expressed in the plant.

Commercial traits in plants can be created through the expression ofgenes that alter starch or protein for the production of paper,textiles, ethanol, polymers or other materials with industrial uses.

Means for increasing or inhibiting a protein are well known to thoseskilled in the art and, include, but are not limited to: transgenicexpression, antisense suppression, co-suppression, RNA interference,gene activation or suppression using transcription factors and/orrepressors; mutagenesis including, but not limited to, transposontagging; directed and site-specific mutagenesis, chromosome engineering(see Nobrega et al., Nature 431:988-993(04)), homologous recombination,TILLING (Targeting Induced Local Lesions In Genomes), and biosyntheticcompetition to manipulate the expression of proteins. Many techniquesfor gene silencing are well known to one of skill in the art, includingbut not limited to knock-outs (such as by insertion of a transposableelement such as Mu, Vicki Chandler, The Maize Handbook ch. 118(Springer-Verlag 1994) or other genetic elements such as a FRT, Lox orother site specific integration site; RNA interference (Napoli et al.(1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323, Sharp (1999) GenesDev. 13:139-141, Zamore et al. (2000) Cell 101:25-33; and Montgomery etal. (1998) PNAS USA 95:15502-15507); virus-induced gene silencing(Burton et al. (2000) Plant Cell 12:691-705, and Baulcombe (1999) Curr.Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff etal. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000)Nature 407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman &Sakai (2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al.(1992) EMBO J. 11:1525, and Perriman et al. (1993) Antisense Res. Dev.3:253); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); zinc-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. (See,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829). By “antisense DNA nucleotidesequence” is intended a sequence that is in inverse orientation to the5′-to-3′ normal orientation of that nucleotide sequence. When deliveredinto a plant cell, expression of the antisense DNA sequence preventsnormal expression of the DNA nucleotide sequence for the targeted gene.The antisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the DNA nucleotidesequence for the targeted gene. In this case, production of the nativeprotein encoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the regulatory sequences disclosed herein canbe operably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant.

As noted, other potential approaches to impact expression of proteins inthe plant include traditional co-supression, that is, inhibition ofexpression of an endogenous gene through the expression of an identicalstructural gene or gene fragment introduced through transformation(Goring, D. R., Thomson, L., Rothstein, S. J. 1991. Proc. Natl. Acad.Sci. USA 88:1770-1774 co-suppression; Taylor (1997) Plant Cell 9:1245;Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA91:3490-3496; Finnegan et al. (1994) Bio/Technology 12:883-888; andNeuhuber et al. (1994) Mol. Gen. Genet. 244:230-241)). In one example,co-suppression can be achieved by linking the promoter to a DNA segmentsuch that transcripts of the segment are produced in the senseorientation and where the transcripts have at least 65% sequenceidentity to transcripts of the endogenous gene of interest, therebysuppressing expression of the endogenous gene in said plant cell. (See,U.S. Pat. No. 5,283,184). The endogenous gene targeted forco-suppression may be a gene encoding any protein that accumulates inthe plant species of interest. For example, where the endogenous genetargeted for co-suppression is the 50 kD gamma-zein gene, co-suppressionis achieved using an expression cassette comprising the 50 kD gamma-zeingene sequence, or variant or fragment thereof.

Additional methods of down-regulation are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see Smithet al. (2000) Nature 407:319-320, Waterhouse and Helliwell (2003)) Nat.Rev. Genet. 4:29-38; Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA95:13959-13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA97:4985-4990; Stoutjesdijk et al. (2002) Plant Phystiol. 129:1723-1731;and Patent Application WO 99/53050; WO 99/49029; WO 99/61631; WO00/49035 and U.S. Pat. No. 6,506,559.

For mRNA interference, the expression cassette is designed to express anRNA molecule that is modeled on an endogenous miRNA gene. The miRNA geneencodes an RNA that forms a hairpin structure containing a 22-nucleotidesequence that is complementary to another endogenous gene (targetsequence). miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants.

In one embodiment, the polynucleotide to be introduced into the plantcomprises an inhibitory sequence that encodes a zinc finger protein thatbinds to a gene encoding a protein of the invention resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a gene of the invention. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding a protein and prevents its translation. Methods of selectingsites for targeting by zinc finger proteins have been described, forexample, in U.S. Pat. No. 6,453,242, and methods for using zinc fingerproteins to inhibit the expression of genes in plants are described, forexample, in U.S. Patent Publication No. 2003/0037355.

The regulatory region of the invention may also be used in conjunctionwith another promoter. In one embodiment, the plant selection marker andthe gene of interest can be both functionally linked to the samepromoter. In another embodiment, the plant selection marker and the geneof interest can be functionally linked to different promoters. In yetother embodiments, the expression vector can contain two or more genesof interest that can be linked to the same promoter or differentpromoters. For example, the SB-OLE promoter described here can be usedto drive the gene of interest and the selectable marker, or a differentpromoter used for one or the other. These other promoter elements can bethose that are constitutive or sufficient to render promoter-dependentgene expression controllable as being cell-type specific,tissue-specific or time or developmental stage specific, or beinginducible by external signals or agents. Such elements may be located inthe 5′ or 3′ regions of the gene. Although the additional promoter maybe the endogenous promoter of a structural gene of interest, thepromoter can also be a foreign regulatory sequence. Promoter elementsemployed to control expression of product proteins and the selectiongene can be any plant-compatible promoters. These can be plant genepromoters, such as, for example, the ubiquitin promoter (Christensen etal. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992)Plant Mol. Biol. 18:675-689); the promoter for the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al, 1984;Broglie et al, 1984); or promoters from the tumor-inducing plasmids fromAgrobacterium tumefaciens, such as the nopaline synthase, octopinesynthase and mannopine synthase promoters (Velten, J. and Schell, J.(1985) “Selection-expression plasmid vectors for use in genetictransformation of higher plants” Nucleic Acids Res. 13:6981-6998;Depicker et al., (1982) Mol. and Appl. Genet. 1:561-573 Shaw et al.(1984) Nucleic Acids Research vol. 12, No. 20, pp. 7831-7846) that haveplant activity; or viral promoters such as the cauliflower mosaic virus(CaMV) 19S and 35S promoters (Guilley et al. (1982) “Transcription ofCauliflower mosaic virus DNA: detection of promoter sequences, andcharacterization of transcripts” Cell 30:763-773; Odell et al. (1985)“Identification of DNA sequences required for activity of thecauliflower mosaic virus 35S promoter” Nature 313:810-812, the figwortmosaic virus FLt promoter (Maiti et al. (1997) “Promoter/leader deletionanalysis and plant expression vectors with the figwort mosaic virus(FMV) full length transcript (FLt) promoter containing single or doubleenhancer domains” Transgenic Res. 6:143-156) or the coat proteinpromoter of TMV (Grdzelishvili et al., 2000) “Mapping of the tobaccomosaic virus movement protein and coat protein subgenomic RNA promotersin vivo” Virology 275, 177-192).

The expression cassette may also include at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source. Thus, any convenient terminationregions can be used in conjunction with the promoter of the invention,and are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also:Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987)Nucleic Acid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al. (1986); MDMV leader (Maize Dwarf Mosaic Virus), Virology154:9-20; human immunoglobulin heavy-chain binding protein (BiP),Macejak et al. (1991) Nature 353:90-94; untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV), Gallie etal. (1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV), Lommel et al. (1991) Virology 81:382-385.See also Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have an expressed product ofan isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella et al.(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983)Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian etal. (1995) Plant Science 108:219-227; streptomycin, Jones et al. (1987)Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard et al.(1996) Transgenic Res. 5:131-137; bleomycin, Hille et al. (1990) PlantMol. Biol. 7:171-176; sulfonamide, Guerineau et al. (1990) Plant Mol.Biol. 15:127-136; bromoxynil, Stalker et al. (1988) Science 242:419-423;glyphosate, Shaw et al. (1986) Science 233:478-481; phosphinothricin,DeBlock et al. (1987) EMBO J. 6:2513-2518.

Further, when linking a promoter of the invention with a nucleotidesequence encoding a detectable protein, expression of a linked sequencecan be tracked in the plant, thereby providing useful so-calledscreenable or scorable markers. The expression of the linked protein canbe detected without the necessity of destroying tissue. By way ofexample without limitation, the promoter can be linked with detectablemarkers including a β-glucuronidase, or uidA gene (GUS), which encodesan enzyme for which various chromogenic substrates are known (Jeffersonet al., 1986, Proc. Natl. Acad. Sci. USA 83:8447-8451); chloramphenicolacetyl transferase; alkaline phosphatase; a R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., in Chromosome Structure andFunction, Kluwer Academic Publishers, Appels and Gustafson eds., pp.263-282 (1988); Ludwig et al. (1990) Science 247:449); a p-lactamasegene (Sutcliffe, Proc. Nat'l. Acad. Sci. U.S.A. 75:3737 (1978)), whichencodes an enzyme for which various chromogenic substrates are known(e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky etal., Proc. Nat'l. Acad. Sci. U.S.A. 80:1101 (1983)), which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., Biotech. 8:241 (1990)); a tyrosinase gene(Katz et al., J. Gen. Microbiol. 129:2703 (1983)), which encodes anenzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which inturn condenses to form the easily detectable compound melanin a greenfluorescent protein (GFP) gene (Sheen et al., Plant J. 8(5):777-84(1995)); a lux gene, which encodes a luciferase, the presence of whichmay be detected using, for example, X-ray film, scintillation counting,fluorescent spectrophotometry, low-light video cameras, photon countingcameras or multiwell luminometry (Teeri et al. (1989) EMBO J. 8:343);DS-RED EXPRESS (Matz et al. (1999) Nature Biotech. 17:969-973, Bevis etal. (2002) Nature Biotech 20:83-87, Haas et al. (1996) Curr. Biol.6:315-324); Zoanthus sp. yellow fluorescent protein (ZsYellow) that hasbeen engineered for brighter fluorescence (Matz et al. (1999) NatureBiotech. 17:969-973, available from BD Biosciences Clontech, Palo Alto,Calif., USA, catalog no. 632443); and cyan florescent protein (CYP)(Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002)Plant Physiol 129:913-42).

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook et al. (supra). The transformation vectorcomprising the regulatory sequence of the present invention operablylinked to an isolated nucleotide sequence in an expression cassette, canalso contain at least one additional nucleotide sequence for a gene tobe cotransformed into the organism. Alternatively, the additionalsequence(s) can be provided on another transformation vector. Vectorsthat are functional in plants can be binary plasmids derived fromAgrobacterium. Such vectors are capable of transforming plant cells.These vectors contain left and right border sequences that are requiredfor integration into the host (plant) chromosome. At minimum, betweenthese border sequences is the gene to be expressed under control of theregulatory elements of the present invention. In one embodiment, aselectable marker and a reporter gene are also included.

A transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated nucleotidesequence of interest in an expression cassette, can be used to transformany plant. In this manner, genetically modified plants, plant cells,plant tissue, and the like can be obtained. Transformation protocols canvary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of transformingplant cells include microinjection, Crossway et al. (1986) Biotechniques4:320-334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83:5602-5606; Agrobacterium-mediated transformation, see forexample, Townsend et al. U.S. Pat. No. 5,563,055; direct gene transfer,Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particleacceleration, see for example, Sanford et al. U.S. Pat. No. 4,945,050,Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);and McCabe et al. (1988) Biotechnology 6:923-926. Also see Weissinger etal. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Bio/Technology8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839; Hooydaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad.Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in TheExperimental Manipulation of Ovule Tissues, ed. G. P. Chapman et al.(Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant CellReports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou et al. (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens).

The cells that have been transformed can be grown into plants inaccordance with conventional methods. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants can then be grown andpollinated with the same transformed strain or different strains. Theresulting plant having constitutive expression of the desired phenotypiccharacteristic can then be identified. Two or more generations can begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Isolation of Regulatory Sequences

Regulatory regions from sorghum SB-OLE (Sorghum bicolor oleosin protein)were isolated from sorghum plants and cloned. Sorghum SB-OLE wasselected as a source of seed preferred regulatory elements based on thespatial and temporal expression of its products.

The promoter is a 948 base pair nucleotide sequence. A BLAST of GenBankshowed the highest identity was 59% to GenBank accession number U13701(GI:687244) by Lee and Huang, “Zea mays oil body protein 16 kDa oleosin(ole16) gene, complete cds” 1995; and Lee and Huang (1994), supra. Aregion of 433 base pairs in the 3′ region of the sequence indicates 78%identity. A comparison of the Zea mays oleosin promoter (SEQ ID NO: 3,the Sorghum oleosin promoter (SEQ ID NO: 1) and the Oryza sativa (SEQ IDNO: 2) oleosin promoter shows common to all three oleosin promoters,thus indicating the importance of these regions. These include:

CCCTCT---TCTCCCT---CCTCAC CGCATGCG-CCACGC CAGCGGC GCCCAC GCCTCCCTCGTA-TATCGCC GCGGCG

The method for isolation of the above sequences is described below.

SB-OLE PRO PCR Product of length 1126 (rating: 171) TMS1615:GAGCTTCACCAAACATGCCC (SEQ ID NO: 4) TMS1616: GACAGCACCAGCATCGACCC (SEQID NO: 5)

Using the maize 16 KD Oleosin transcript, the sorghum ortholog wasidentified and used to identify genomic sequences containing thepromoter from the NCBI GSS (Genome Survey Sequence) collection. Thepromoter was amplified from sorghum line genomic DNA using PCR. The PCRreaction was performed in a Bio-Rad icycler (Hercules, Calif.) thermalcycler using Hifidelity supermix (Cat. # 10790-020, Life Technologies,Rockville Md.). The following cycle parameters were used: 94° C. for 2seconds, followed by 30 cycles of 94° C. for 20 seconds, for 30 seconds,and 68° C. for 1 minute. Finally, the samples were held at 67° C. for 4minutes and then at 4° C. until further analysis.

The PCR products were cloned into the (Promega™) pGEM-easy vector andsequenced. Upon sequence verification, they were given PHP numbers andarchived. Once amplified, the PCR fragments were sequenced and assembledinto expression cassettes using the YELLOW FLUORESCENT PROTEIN (YFP)coding region (supra) as the marker gene.

Example 2 Expression Data Using Promoter Sequences

A construct, named PHP28986, was prepared which included the sorghumoleosin promoter (SEQ ID NO:1) linked with the YFP selectable marker,supra and the nos or nopaline synthase transcription terminator(Depicker et al. (1982) J. Mol. Appl. Genet. 1 (6):561-573; Shaw et al.(1984) Nucleic Acids Research Vol. 12, No. 20, pp. 7831-7846). Allvectors were constructed using standard molecular biology techniques(Sambrook et al., supra). Successful subcloning was confirmed byrestriction analysis.

Example 3 Transformation and Regeneration of Maize Callus ViaAgrobacterium

Constructs used were as those set forth supra using a binary plasmidwith the left and right borders (see Hiei et al., U.S. Pat. No.7,060,876) and the selectable marker for maize-optimized PAT(phosphinothricin acetyl transferase). Jayne et al., U.S. Pat. No.6,096,947.

Preparation of Agrobacterium Suspension:

Agrobacterium was streaked out from a −80° frozen aliquot onto a platecontaining PHI-L medium and was cultured at 28° C. in the dark for 3days. PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l StockSolution B, 450.9 ml/l Stock Solution C and spectinomycin (SigmaChemicals) was added to a concentration of 50 mg/l in sterile ddH2O(stock solution A: K2HPO4 60.0 g/l, NaH2PO4 20.0 g/l, adjust pH to 7.0w/KOH and autoclaved; stock solution B: NH4Cl 20.0 g/l, MgSO4.7H2O 6.0g/l, KCl 3.0 g/l, CaCl2 0.20 g/l, FeSO4.7H2O 50.0 mg/l, autoclaved;stock solution C: glucose 5.56 g/l, agar 16.67 g/l (#A-7049, SigmaChemicals, St. Louis, Mo.) and was autoclaved).

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony was picked from the master plate and was streaked onto aplate containing PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco) 10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8,containing 50 mg/L spectinomycin] and was incubated at 28° C. in thedark for 2 days.

Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l,Eriksson's vitamin mix (1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l;L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l; glucose(Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic medium system, orPHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma) 0.5 mg/l;pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol(Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab) 1 g/l; 2,4-D1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOHand filter-sterilize] for the PHI combined medium system and 5 ml of 100mM (3′-5′-Dimethoxy-4′-hydroxyacetophenone, Aldrich chemicals) was addedto a 14 ml Falcon tube in a hood. About 3 full loops (5 mm loop size)Agrobacterium was collected from the plate and suspended in the tube,then the tube vortexed to make an even suspension. One ml of thesuspension was transferred to a spectrophotometer tube and the OD of thesuspension is adjusted to 0.72 at 550 nm by adding either moreAgrobacterium or more of the same suspension medium, for anAgrobacterium concentration of approximately 0.5×109 cfu/ml to 1×109cfu/ml. The final Agrobacterium suspension was aliquoted into 2 mlmicrocentrifuge tubes, each containing 1 ml of the suspension. Thesuspensions were then used as soon as possible.

Embryo Isolation, Infection and Co-Cultivation:

About 2 ml of the same medium (here PHI-A or PHI-I) which is used forthe Agrobacterium suspension was added into a 2 ml microcentrifuge tube.Immature embryos were isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos was about 1.0-1.2 mm. The cap was thenclosed on the tube and the tube vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium wasremoved and 2 ml of fresh medium were added and the vortexing repeated.All of the medium was drawn off and 1 ml of Agrobacterium suspension wasadded to the embryos and the tube is vortexed for 30 sec. The tube wasallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos was poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; gelrite (Sigma) 3.0 g/l;sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the PHI basicmedium system, or PHI-J medium [MS Salts 4.3 g/l; nicotinic acid 0.50mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol100.0 mg/l; 2,4-D 1.5 mg/l; sucrose 20.0 g/l; glucose 10.0 g/l;L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (Sigma A-7049,purified) and 100 mM acetosyringone with a final pH of 5.8 for the PHIcombined medium system. Any embryos left in the tube were transferred tothe plate using a sterile spatula. The Agrobacterium suspension wasdrawn off and the embryos placed axis side down on the media. The platewas sealed with Parafilm tape or Pylon Vegetative Combine Tape (productnamed “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd.,Japan) and was incubated in the dark at 23-25° C. for about 3 days ofco-cultivation.

Resting, Selection and Regeneration Steps:

For the resting step, all of the embryos were transferred to a new platecontaining PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l;Eriksson's vitamin mix (1000× Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver nitrate0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate was sealed withParafilm or Pylon tape and incubated in the dark at 28° C. for 3-5 days.

Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation

For selection, all of the embryos were then transferred from the PHI-Cmedium to new plates containing PHI-D medium, as a selection medium,[CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitamin mix(1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K.K., Tokyo, Japan)1.5 mg/l for the first two weeks followed by 3 mg/l for the remainder ofthe time; pH 5.8] putting about 20 embryos onto each plate.

The plates were sealed as described above and incubated in the dark at28° C. for the first two weeks of selection. The embryos weretransferred to fresh selection medium at two-week intervals. The tissuewas subcultured by transferring to fresh selection medium for a total ofabout 2 months. The herbicide-resistant calli are then “bulked up” bygrowing on the same medium for another two weeks until the diameter ofthe calli is about 1.5-2 cm.

For regeneration, the calli were then cultured on PHI-E medium [MS salts4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l, thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin 0.5 mg/l,sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleacetic acid (IAA,Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos 3 mg/l,carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3weeks to allow somatic embryos to mature. The calli were then culturedon PHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5mg/l; sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at 25° C. under adaylight schedule of 16 hrs. light (270 uE m−2sec−1) and 8 hrs. darkuntil shoots and roots are developed. Each small plantlet was thentransferred to a 25×150 mm tube containing PHI-F medium and is grownunder the same conditions for approximately another week. The plantswere transplanted to pots with soil mixture in a greenhouse. YFP eventswere determined at regenerated plant stage.

Ability of the SB-OLE promoter to drive expression in maize wasconfirmed by YFP detection in plant tissue by the procedures outlinedsupra. It was determined that the SB-OLE PRO drove expression in maizesimilar to that driven by the maize 16 KD Oleosin promoter. YFP wasdetected in the embryo and aleurone from about 12 DAP through seedmaturity. No leaf expression was observed.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited are incorporate herein by reference.

1. A method for selectively expressing a nucleotide sequence in a plantcell, the method comprising: a) transforming a plant cell with anexpression cassette, the expression cassette comprising a promoteroperably linked to a nucleotide sequence wherein the promoter comprisesa nucleotide sequence selected from the group consisting of: i) thenucleotide sequence set forth in SEQ ID NO: 1 or 2; and ii) a functionalfragment of i); and b) growing the plant cell to selectively express thenucleotide sequence.
 2. The method of claim 1 wherein the promoterinitiates expression of the nucleotide sequence in seed embryo/aleuronetissue.
 3. The method of claim 1 further comprising regenerating astably transformed plant from the plant cell; wherein expression of thenucleotide sequence alters the phenotype of a plant.
 4. The plant ofclaim 3, wherein said plant is a monocot.
 5. The plant of claim 3,wherein said monocot is maize, wheat, rice, barley, sorghum, or rye. 6.Seed of the plant of claim 3 wherein the seed comprises the expressioncassette.